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Genotyping Citrus tristeza virus Isolates by Sequential Multiplex RT-PCR and Microarray Hybridization in a Lab-on-Chip Device

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Citrus Tristeza Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2015))

Abstract

Citrus tristeza virus (CTV) is the largest known plant RNA virus (ca. 20 Kb), with a plethora of isolates conventionally categorized into six main genotypic groups (T36, VT, T3, RB, T68, T30). Each group includes many isolates with different phenotype profiles. Several techniques and protocols, mostly based on RT-PCR analysis of different regions of specific genes, have been developed for managing the diseases caused by CTV. However, more accurate genomic information would help to plan a correct strategy. This chapter describes a pilot protocol based on a sequential multiplex RT-PCR reaction and microarray hybridization in a miniaturized silicon lab-on-chip (LoC) device. The system comprises a set of 12 primers and 44 probes (× 2 replicates), designed on variable genomic regions of 6 genes: 5′UTR, ORF1a, ORF1b (RdRp), p33, p20, and p23. The system can rapidly analyze any genotype diversity associated with field isolates and distinguish the endemic from the non-endemic isolates. The identification of CTV strains is based on a number of probe hybridizations, which varies according to the genotypes present in the isolates and the differences among the genotypes.

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Acknowledgments

This work was funded in part by the MIUR and EU through the National Program PON R&C 2007–2013, project “IT-Citrus genomics” (PON 01_01623), led by Science and Technology Park of Sicily, Catania (Italy).

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Authors and Affiliations

  1. Agrobiotech Soc. Coop., Catania, Italy

    Giuseppe Scuderi

  2. Formerly, Department of Phytosanitary Sciences, University of Catania, Catania, Italy

    Antonino F. Catara

  3. Science and Technology Park of Sicily, Catania, Italy

    Antonino F. Catara

  4. Consiglio per la Ricerca in agricoltura e l’analisi dell’Economia Agraria (CREA), Centro di Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA), Acireale (Catania), Italy

    Grazia Licciardello

Authors
  1. Giuseppe Scuderi
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  2. Antonino F. Catara
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  3. Grazia Licciardello
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Corresponding author

Correspondence to Giuseppe Scuderi .

Editor information

Editors and Affiliations

  1. Department of Phytosanitary Sciences, University of Catania, Science and Technology Park of Sicily, Catania, Italy

    Antonino F. Catara

  2. The S. Talkowski Laboratory, Department of Plant Pathology and Weed Research, The Volcani Center, Agricultural Research Organization, Bet Dagan, Israel

    Moshe Bar-Joseph

  3. Consiglio per la Ricerca in agricoltura e l’analisi dell’Economia Agraria, Centro di Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA), Acireale (Catania), Italy

    Grazia Licciardello

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Scuderi, G., Catara, A.F., Licciardello, G. (2019). Genotyping Citrus tristeza virus Isolates by Sequential Multiplex RT-PCR and Microarray Hybridization in a Lab-on-Chip Device. In: Catara, A., Bar-Joseph, M., Licciardello, G. (eds) Citrus Tristeza Virus. Methods in Molecular Biology, vol 2015. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9558-5_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9558-5_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9557-8

  • Online ISBN: 978-1-4939-9558-5

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